The assembly and release of HIV-1 particles is quite complex, in that a number of viral and cellular RNAs and proteins participate, and while much has been learned about this process, much remains to be discovered. The Gag protein is central to the assembly and release of HIV-1 and all other retrovirus particles. A key function of the Gag protein that will be one focus of tis proposal is the recruitment and packaging of viral genomic RNA (gRNA). Reciprocally, gRNA may help to drive the recruitment of additional Gag molecules into a virion as assembly progresses. However, the full extent to which RNA participates in particle assembly in cells, and how Gag:RNA interactions change as assembly is initiated and completed remain to be defined. The experiments proposed in specific aim 1 will be the first to determine precisely how HIV-1 Gag and gRNA interact in cells. These studies could identify the very first as well as subsequent interactions between Gag protein and gRNA as well as between gRNA and antiviral cytidine deaminases. Following assembly, HIV-1 particles depart from the surface of the infected cell, to colonize neighboring or distal uninfected cells. During the previous funding period, we identified an IFN-induced membrane protein, termed tetherin, as a host defense molecule that inhibits HIV-1 particle release. Our work has demonstrated that tetherin acts directly, to retain nascent virions on the cell surface, but key mechanistic details of how tetherin functions, and what role t plays in preventing the dissemination of viral infection in cultured cells and in vivo remain to b determined. In specific aim 2, we will perform a series of in vitro and in vivo experiments to address these key questions. PUBLIC HEALTH RELEVANCE: The genesis of infectious extracellular HIV-1 particles is essential for the propagation of infection between individual cells and hosts. Understanding HIV-1 particle assembly and release may offer a range of opportunities for intervention as a therapeutic strategy. This proposal seeks to understand how HIV-1 genetic material is packaged into virions and how antiviral proteins interfere with the genesis of infectious particles.